Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Inhibition of bfl-1/A1 by siRNA inhibits mycobacterial growth in THP-1 cells by enhancing phagosomal acidification

Virulent tubercle bacilli inhibit apoptosis to establish a safe environment within the host cells. Here, we report that NF-kappaB dependent antiapoptotic protein bfl-1/A1 plays an important role in this process. Both virulent and avirulent mycobacteria bearing THP-1 cells expressed considerable amount of bfl-1/A1 after 4 h of infection. However, after 48 h of infection, bfl-1/A1 expression was evident only in Mycobacterium tuberculosis H37Rv but not in M. tuberculosis H37Ra infected cells. When parallel experiments were performed with Human monocyte-derived macrophages (MDMs), differential expression of bfl-1/A1 mRNA was observed in case of M. tuberculosis H37Rv and M. tuberculosis H37Ra infection. siRNA mediated inhibition of bfl-1/A1 induced apoptosis in M. tuberculosis H37Rv infected THP-1 and MDMs. Reduction in intracellular mycobacterial growth was observed in bfl-1/A1 siRNA transfected, M. tuberculosis H37Rv infected THP-1 cells. Enhancement of phagosome-lysosome fusion was observed in bfl-1/A1 siRNA treated and M. tuberculosis H37Rv infected THP-1 cells. These results clearly indicated that differential expression of bfl-1/A1 in M. tuberculosis H37Rv and M. tuberculosis H37Ra infected THP-1 cells probably account for the difference in infection outcome.     

Survival of Mycobacterium tuberculosis in host macrophages involves resistance to apoptosis dependent upon induction of antiapoptotic Bcl-2 family member Mcl-1

Mcl-1 protein expression was found to be up-regulated during infection with virulent Mycobacterium tuberculosis strain H37Rv. Mcl-1 induction in THP-1 cells was optimal at a multiplicity of infection of 0.8-1.2 bacilli per macrophage and was independent of opsonin coating of the bacteria. Mcl-1 expression was elevated as early as 4 h, peaked at 5.8-fold above control cells at 24 h, and remained elevated at 48 h after infection. In THP-1 cells, mMcl-1 mRNA was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. In THP-1 cells and monocyte-derived macrophages (MDMs), Mcl-1 protein was induced by infection with live H37Rv but not with attenuated M. tuberculosis strain H37Ra, heat-killed H37Rv, or latex beads. Treatment of uninfected, H37Ra-infected, and H37Rv-infected THP-1 cells and MDMs with antisense oligonucleotides to mcl-1 reduced Mcl-1 expression by >84%. This resulted in an increase in apoptosis of both MDMs and THP-1 cells that were infected with H37Rv, but not cells that were uninfected or infected with H37Ra. Increased apoptosis correlated with a decrease in M. tuberculosis CFUs recovered from antisense-treated, H37Rv-infected cells at 4 and 7 days after infection. In contrast, CFU recoveries from sense-treated, H37Rv-infected cells or from antisense- or sense-treated, H37Ra-infected cells were unchanged from controls. Thus, the antiapoptotic effect of the induction of Mcl-1 expression in H37Rv-infected macrophages promotes the survival of virulent M. tuberculosis.     

Survival of virulent Mycobacterium tuberculosis involves preventing apoptosis induced by Bcl-2 upregulation and release resulting from necrosis in J774 macrophages

Macrophage apoptosis plays a role in mycobacterial infection. To define the mechanism by which virulent Mycobacterium tuberculosis escapes apoptosis and killing in macrophages, J774 macrophages were infected with virulent M. tuberculosis H37Rv and attenuated H37Ra strains. H37Rv induced less apoptosis than H37Ra, and caspase 3 was activated in H37Ra- and H37Rv-infected macrophages. Intracellular H37Rv bacilli were released at a higher rate into the supernatant than were H37Ra by the sixth day of infection, and this was simultaneously accompanied by the increased necrosis of infected cells showing lactate dehydrogenase (LDH) release. Fas mRNA expression was downregulated and FasL was upregulated in H37Ra- and H37Rv-infected macrophages, while Bcl-2 was upregulated in H37Rv-infected macrophages but downregulated in H37Ra-infected macrophages as seen by real-time PCR. These results indicate that M. tuberculosis H37Ra and H37Rv proliferate in macrophages by preventing them from inducing apoptosis during the early phase of infection, and that M. tuberculosis H37Rv-infected macrophages are found to express Bcl-2 mRNA, which leads to anti-apoptotic activity, and that relatively distinct necrosis might occur during the later phase of infection.     

Requirement of gene fadD33 for the growth of Mycobacterium tuberculosis in a hepatocyte cell line

Gene fadD33 of Mycobacterium tuberculosis, one of the 36 homologues of gene fadD of Escherichia coli identified in the M. tuberculosis genome, predictively encodes an acyl-CoA synthase, an enzyme involved in fatty acids metabolism. The gene is underexpressed in the attenuated strain M. tuberculosis H37Ra relative to virulent H37Rv and plays a role in M. tuberculosis virulence in BALB/c mice by supporting mycobacterial replication in the liver. In the present paper, we investigated the role of fadD33 expression in bacterial growth within the hepatocyte cell line HepG2, as well as in human monocyte-derived THP-1 cells and peripheral blood mononuclear cells. M. tuberculosis H37Rv proved able to grow within HepG2 cells, while the intracellular replication of M. tuberculosis H37Ra was markedly impaired; complementation of strain H37Ra with gene fadD33 restored its replication to the levels of H37Rv. Moreover, disruption of gene fadD33 by allelic exchange mutagenesis reduced the intracellular growth of M. tuberculosis H37Rv, and complementation of the fadD33-disrupted mutant with gene fadD33 restored bacterial replication. Conversely, fadD33 expression proved unable to influence M. tuberculosis growth in human phagocytes, as fadD33-disrupted M. tuberculosis H37Rv mutant, as well as fadD33-complemented M. tuberculosis H37Ra, grew within THP-1 cells and peripheral monocytes basically at the same rates as parent H37Rv and H37Ra strains. The results of these experiments indicate that gene fadD33 expression confers growth advantage to M. tuberculosis in immortalized hepatocytes, but not in macrophages, thus emphasizing the importance of fadD33 in liver-specific replication of M. tuberculosis.     

Bfl-1/A1 acts as a negative regulator of autophagy in mycobacteria infected macrophages

Expression of Bcl-2 family protein, Bfl-1/A1 has been found to differ considerably amongst macrophages infected with virulent Mycobacterium tuberculosis H37Rv or with avirulent M. tuberculosis H37Ra. Present work was undertaken to deduce the significance of differential expression of Bfl-1/A1 in the outcome of mycobacterial infection. We have studied the role of Bfl-1/A1 particularly in autophagy formation in tubercle bacilli infected cells since autophagy has been recognized as a component of innate immunity against pathogenic mycobacteria. First, we have confirmed that upon infection virulent strain H37Rv retain Bfl-1/A1 for longer period and impose autophagosome maturation block within infected cells as evident from confocal microscopy. Moreover, down regulation of Bfl-1/A1 by siRNA induced autophagy formation and reduced bacterial growth. Furthermore, even the avirulent strain H37Ra resist autophagosome maturation and survive if the cellular level of Bfl-1 is maintained in THP-1 cells by stable transfection (Bfl-1 overexpressing cells). No noteworthy difference in mTOR expression was observed between normal THP-1 and Bfl-1 overexpressing THP-1 cells infected with either strain of mycobacteria. Interestingly, we found that not only mTOR but also Bfl-1/A1 is involved in rapamycin induced autophagy in mycobacteria infected macrophages. We have found that Bfl-1 physically interacts with Beclin 1 in Bfl-1 overexpressing THP-1 as well as in H37Rv infected THP-1 cells as they co-precipitated. Taken together, our results clearly demonstrated that Bfl-1/A1 negatively regulates autophagy and expression of Bfl-1/A1 in H37Rv infected macrophages provides the bacteria a survival strategy to overcome host defense.     

Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages

Human alveolar macrophages (AMphi) undergo apoptosis following infection with Mycobacterium tuberculosis in vitro. Apoptosis of cells infected with intracellular pathogens may benefit the host by eliminating a supportive environment for bacterial growth. The present study compared AMphi apoptosis following infection by M. tuberculosis complex strains of differing virulence and by Mycobacterium kansasii. Avirulent or attenuated bacilli (M. tuberculosis H37Ra, Mycobacterium bovis bacillus Calmette-Guérin, and M. kansasii) induced significantly more AMphi apoptosis than virulent strains (M. tuberculosis H37Rv, Erdman, M. tuberculosis clinical isolate BMC 96.1, and M. bovis wild type). Increased apoptosis was not due to greater intracellular bacterial replication because virulent strains grew more rapidly in AMphi than attenuated strains despite causing less apoptosis. These findings suggest the existence of mycobacterial virulence determinants that modulate the apoptotic response of AMphi to intracellular infection and support the hypothesis that macrophage apoptosis contributes to innate host defense in tuberculosis.     

Comparative proteome analysis of culture supernatant proteins of Mycobacterium tuberculosis H37Rv and H37Ra

To examine the virulence factors of Mycobacterium tuberculosis H37Rv, the proteome was used to characterize the differences in protein expression between virulent M. tuberculosis H37Rv and attenuated M. tuberculosis H37Ra. Two-dimensional gel electrophoresis was performed to separate culture supernatant proteins extracted from M. tuberculosis H37Rv and M. tuberculosis H37Ra. The protein spots of interest were identified by mass spectrometry, and then the genes encoding the identified proteins were cloned and sequenced. Comparison of silver-stained gels showed that three well-resolved protein spots were present in M. tuberculosis H37Rv but absent from M. tuberculosis H37Ra. Protein spot no. 1 was identified as Rv2346c. Protein spot no. 2 was identified as Rv2347c, Rv1197, Rv1038c, and Rv3620c, which shared significant homology and had the same peptide fingerprinting using tryptic digestion. No M. tuberculosis protein matched protein spot no. 3. Rv2346c, Rv2347c, Rv1038c, and Rv3620c of M. tuberculosis H37Rv were located on the M. tuberculosis H37Ra chromosome, and multiple mutations were observed in the corresponding areas of M. tuberculosis H37Ra. Codon 59 (CAG, Gln) of Rv2347c and Rv3620c was replaced by termination codon (TAG) in M. tuberculosis H37Ra, which probably terminated the polypeptide elongation. These results demonstrate the importance of studying the gene products of M. tuberculosis and show that subtle differences in isogenic mutant strains might play an important role in identifying the attenuating mutations.     

Mycobacterium tuberculosis H37Rv induces monocytic release of interleukin-6 via activation of mitogen-activated protein kinases: inhibition by N-acetyl-L-cysteine

The release of proinflammatory cytokines after mycobacterial infection is a host immune response that may be propitious or deleterious to the host. Elevated levels of interleukin (IL)-6 are present in plasma of patients with active tuberculosis infection. The aim of this study was to investigate the role of mitogen-activated protein kinases in the secretion of interleukin-6 in THP-1 cells and human primary monocytes that were infected with Mycobacterium tuberculosis H37Rv, and its regulation by N-acetyl-L-cysteine, a potential antimycobacterial agent. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv induced rapidly, in a time-dependent manner, the phosphorylation of mitogen-activated protein kinase kinase 3/6 and p38 mitogen-activated protein kinase, accompanied by an upregulation of interleukin-6. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and nuclear factor-kappaB, we found that extracellular-signal regulated kinase 1/2, p38 mitogen-activated protein kinase and nuclear factor-kappaB were essential for M. tuberculosis H37Rv-induced interleukin-6 production in human primary monocytes. Pretreatment with N-acetyl-L-cysteine reduced, in a dose-dependent manner, M. tuberculosis H37Rv-induced activation of mitogen-activated protein kinase kinase 3/6 and interleukin-6 production in THP-1 cells.     

Differential release of tumor necrosis factor-α from murine peritoneal macrophages stimulated with virulent and avirulent species of mycobacteria

Abstract The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-α (TNF-α) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-α compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-α; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-α secretion. The TNF-α response was dose-dependent, related directly to the percentage of infected cells, and peaked 6–12 h post-infection. An early and vigorous TNF-α response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.     

Genes of Mycobacterium tuberculosis H37Rv downregulated in the attenuated strain H37Ra are restricted to M. tuberculosis complex species

By comparing gene expression of virulent Mycobacterium tuberculosis H37Rv and attenuated strain H37Ra, we previously detected six genes that appear to be markedly downregulated in the attenuated strain compared with the virulent one. Three of these genes, i.e. Rv1345, Rv2770c, and Rv0288, code for proteins that can be predictively associated to immunological or pathogenetic aspects of M. tuberculosis infection; the other genes, i.e. Rv2336, Rv1320c, and Rv2819c, code for proteins with unknown functions (Rindi et al., 1999). In this paper we searched for the above mentioned genes in Pvu II-digested genomic DNA of a number of mycobacterial species by southern blot analysis employing PCR-generated probes in high-stringency conditions. Hybridization signals were only found in species belonging to the M. tuberculosis complex, i.e., M. tuberculosis, M. bovis, including the BCG strain, and M. microti, but not in other mycobacterial species, including M. avium, M. intracellulare, M. malmoense, M. xenopi, M. kansasii, M. simiae, M. marinum, M. scrofulaceum, M. gordonae, M. fortuitum, and M. smegmantis. These results indicate that genes Rv1345, Rv2770c, Rv0288, Rv2336, Rv1320c, and Rv2819c are associated with the most virulent mycobacteria and further support their potential role in M. tuberculosis virulence.     

Mitogen-activated protein kinases mediate <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>–induced CD44 surface expression in monocytes

CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.     

利用表型芯片系统高通量分析H37Ra与H37Rv差异表型

H37Rv是结核分枝杆菌标准有毒株,H37Ra是从H37Rv获得的稳定减毒株,但目前H37Ra毒力减弱原因尚不完全清楚。本研究利用表型芯片系统,高通量分析H37Ra生长表型,并与H37Rv表型比较,筛选两菌株表型差异,分析与H37Ra毒力减弱可能的相关表型及分子机制。结果发现,与H37Rv相比,H37Ra耐酸及耐渗透压能力显著下降,且不能利用丁二酸单甲酯和吐温40作为碳源。结核分枝杆菌耐酸能力直接影响其在吞噬体中的生存和代谢,耐高渗能力影响其必需营养物质的跨膜运输,代谢途径的改变影响其在宿主内的能量摄取,三者改变均可能与H37Ra毒力减弱相关。     

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Effects of mycobacterial infection on proliferation of hematopoietic precursor cells

Bacterial infection can affect hematopoietic precursor cells in bone marrow, because the infected tissues produce various cytokines and chemokines. Little is known about hematopoietic precursor cells, including hematopoietic stem cells and their progenitors, during mycobacterial infection. Here, we showed that mycobacterial infections result in the expansion of not only the lin-c-kit+sca-1+ (LKS+) cell population, but also granulocyte-monocyte progenitor cells in a chronic murine tuberculosis model. Interestingly, stimulation of LKS+ cells with attenuated Mycobacterium tuberculosis H37Ra culture filtrate (RaCF) was significantly stronger than that by virulent H37Rv culture filtrate (RvCF). Lower TNF-α and IL-6 levels were observed in RvCF-stimulated bone marrow cells. Neutralization of TNF-α or IL-6 in RaCF-stimulated bone marrow cells markedly suppressed LKS+ cell clonal expansion. Additionally, numbers of LKS+ cells were lower in TLR2(-/-) and MyD88(-/-) mice after mycobacterial infection. Taken together, LKS+ cell proliferation related to mycobacterial virulence may be related to the secretion of TNF-α and IL-6 associated with TLR signaling. Expansion of hematopoietic progenitor cells may, therefore, play an important role during mycobacterial infection.     

IL-6 inhibits IFN-γ induced autophagy in Mycobacterium tuberculosis H37Rv infected macrophages

The significance of IL-6 production in tuberculosis is yet to be fully elucidated, although it is known for quite some time that IL-6 interferes with IFN-γ induced signal. In order to know which cellular process induced by IFN-γ is actually counteracted by IL-6, we studied the role of IL-6 on IFN-γ induced autophagy formation in virulent Mycobacterium tuberculosis infection in THP-1 cells, since it is well characterized that induction of autophagy by IFN-γ eliminates intracellular mycobacterium by overcoming the phagosome maturation block imposed by bacilli. We report here that IL-6 inhibits both IFN-γ and starvation induced autophagy in M. tuberculosis H37Rv infected cells. M. tuberculosis H37Rv infection results in time dependent production of IL-6 in THP-1 cells and neutralization of this endogenous IL-6 by anti-IL-6 antibody significantly enhances the IFN-γ mediated killing of the intracellular bacteria. IL-6 time dependently lowers Atg12-Atg5 complex and therefore inhibits autophagosome biogenesis rather than autophagolysosome formation. IL-6 also affects IFN-γ mediated stimulation of mTOR, p-38 and JNK pathways. These results clearly indicate that virulent mycobacteria strategically upregulate IL-6 production to combat innate immunity.     

Deoxyribonucleic acid methylation in mycobacteria.

Deoxyribonucleic acid modification in six strains of mycobacteria was investigated. The presence of 5-methylcytosine in the virulent strain Mycobacterium tuberculosis H37Rv and its absence in the avirulent strain M. tuberculosis H37Ra and other saprophytic, fast-growing mycobacteria appear to be the salient features. However, deoxyribonucleic acid from M. smegmatis SN2 lysogenized with the temperature phage I3 showed the presence of 5-methylcytosine. All of the strains had N6-methyladenine.